Regulation of proliferation and differentiation of respiratory tract epithelial cells by TGFβ

doi:10.1016/0014-4827(86)90193-X

Experimental Cell Research

Volume 167, Issue 2, December 1986, Pages 539-549

https://doi.org/10.1016/0014-4827(86)90193-X Get rights and content

Abstract

In this paper we examined the effects of transforming growth factor β (TGFβ) on the proliferation and differentiation of rabbit tracheal epithelial cells in primary culture. Treatment of these cells with TGFβ inhibits cell proliferation in a time- and dose-dependent manner; concentrations as low as 1 pM are able to inhibit cell growth. Concomitantly, TGFβ causes cells to accumulate in the $G0 G1$ phase of the cell cycle and a sharp reduction in the ability of the cells to form colonies after subculture at clonal density. These results indicate that TGFβ induces terminal cell division in these cells. The inhibition of cell growth is accompanied by changes in cell morphology and a stimulation of the formation of cross-linked envelopes. TGFβ enhances the levels of transglutaminase activity and cholesterol sulfate, two markers of squamous differentiation. Our results indicate that TGFβ induces terminal squamous cell differentiation in rabbit tracheal epithelial cells. Retinoic acid (RA) does not affect the commitment to terminal cell division induced by TGFβ, but inhibits the expression of the squamous phenotype. Growth of normal human bronchial epithelial cells was affected by TGFβ in a way similar to that of rabbit tracheal epithelial cells. Several carcinoma cell lines tested were quite resistant to TGFβ, whereas growth of one carcinoma cell line was stimulated by TGFβ. These results indicate that a modified response to TGFβ could be one mechanism involved in the aberrant growth control of malignant cells.

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Among the many factors that have been identified and regulate positively or negatively the growth and differentiation of airway epithelial cells by autocrine or paracrine mechanisms, transforming growth factor β (TGF-β), a major cytokine involved in driving squamous metaplasia, is of particular interest. Jetten et al.38 and Masui et al.64 have indeed shown that TGF-β exposure or overexpression in airway epithelial cells induces terminal cell division and favors the expression of a squamous phenotype. Similarly in the present study, αBTX-induced hyperproliferation is followed by a period in which cells stop proliferating and undergo squamous differentiation, suggesting that these changes in the differentiation pathway, when α7 nAChR is absent or inactivated, may be related to TGF-β overexpression.
Airway epithelial basal cells are known to be critical for regenerating injured epithelium and maintaining tissue homeostasis. Recent evidence suggests that the α7 nicotinic acetylcholine receptor (nAChR), which is highly permeable to Ca²⁺, is involved in lung morphogenesis. Here, we have investigated the potential role of the α7 nAChR in the regulation of airway epithelial basal cell proliferation and the differentiation of the human airway epithelium. In vivo during fetal development and in vitro during the regeneration of the human airway epithelium, α7 nAChR expression coincides with epithelium differentiation. Inactivating α7 nAChR function in vitro increases cell proliferation during the initial steps of the epithelium regeneration, leading to epithelial alterations such as basal cell hyperplasia and squamous metaplasia, remodeling observed in many bronchopulmonary diseases. The regeneration of the airway epithelium after injury in α7^−/− mice is delayed and characterized by a transient hyperplasia of basal cells. Moreover, 1-year-old α7^−/− mice more frequently present basal cells hyperplasia. Modulating nAChR function or expression shows that only α7 nAChR, as opposed to heteropentameric α_xβ_y nAChRs, controls the proliferation of human airway epithelial basal cells. These findings suggest that α7 nAChR is a key regulator of the plasticity of the human airway epithelium by controlling basal cell proliferation and differentiation pathway and is involved in airway remodeling during bronchopulmonary diseases.
Modulation of the transforming growth factor-β signal transduction pathway by hepatitis C virus nonstructural 5A protein
2006, Journal of Biological Chemistry
Citation Excerpt :
Using a luciferase reporter gene assay, we found that NS5B protein had no effect on the TGF-β signaling pathway (data not shown). TGF-β induces hepatic fibrogenesis and modulates the proliferation and differentiation of epithelial cells (50-52). TGF-β inhibits cell cycle progression during G1 phase through enhanced expression of cyclin-dependent kinase inhibitors such as p21WAF1/CIP1 (53).
Transforming growth factor-β (TGF-β) is implicated in the pathogenesis of liver disease. TGF-β is involved both in liver regeneration and in the fibrotic and cirrhotic transformation with hepatitis viral infection. Hepatitis C virus (HCV) infection often leads to cirrhosis and hepatocellular carcinoma. HCV nonstructural 5A (NS5A) protein is a multifunctional protein that modulates cytokine-mediated signal transduction pathways. To elucidate the molecular mechanism of HCV pathogenesis, we examined the effect of NS5A protein on TGF-β-stimulated signaling cascades. We show that NS5A protein inhibited the TGF-β-mediated signaling pathway in hepatoma cell lines as determined by reporter gene assay. To further investigate the role of NS5A, we examined the protein/protein interaction between NS5A and TGF-β signal transducers. Both in vitro and in vivo binding data showed that NS5A protein directly interacted with TGF-β receptor I (TβR-I) in hepatoma cell lines. This interaction was mapped to amino acids 148-238 of NS5A. We also found that NS5A protein co-localized with TβR-I in the cytoplasm of Huh7 cells and inhibited TGF-β-mediated nuclear translocation of Smad2. Furthermore, we demonstrate that NS5A protein abrogated the phosphorylation of Smad2 and the heterodimerization of Smad3 and Smad4. To further explore the relevance to viral infection, we examined the effect of the HCV subgenomic replicon on the TGF-β signaling pathway. We show that the HCV subgenomic replicon also inhibited TGF-β-induced signaling cascades. These results indicate that HCV NS5A modulates TGF-β signaling through interaction with TβR-I and that NS5A may be an important risk factor in HCV-associated liver pathogenesis.
Gene expression of transforming growth factor β receptors I and II in non-small-cell lung tumors
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Transforming growth factor (TGF)β inhibits normal epithelial cell proliferation. A decreased expression of TGFβ receptors (TβR) has been associated with loss of TGFβ sensitivity and enhanced tumor progression in many types of cancer. Although lung cancer is one of the leading causes of cancer death, a comparative analysis of TβR mRNA and protein expression in non-small-cell (NSC) lung tumors has not been performed. Lung tumor tissues and control non-lesional lung tissues were obtained from 17 patients undergoing thoracotomy for primary NSC lung tumors in clinical stage II. Each tissue sample was studied for TβRI and TβRII mRNA and immunoreactive protein expression, using a semi-quantitative reverse transcription-PCR method, and a quantitative immunohistochemistry method, respectively. TβRI protein expression was higher in tumors than in controls (p=0.0005) and a similar trend was present at the mRNA level. TβRII protein expression was not significantly different between tumors and controls, however an intense peri-nuclear staining for TβRII was observed in several tumor cells. TβRII mRNA levels were lower in tumors than in controls (p=0.005) and an inverse relation between TβRII mRNA and protein expression was detected in tumors (p=0.0013). Our findings suggest an altered function of the TβR system in NSC lung cancer.
Lack of correlation between growth inhibition by TGF-β and the percentage of cells expressing type II TGF-β receptor in human non-small cell lung carcinoma cell lines
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To determine the mechanisms involved in the evasion from TGF-β growth regulation in the small cell lung carcinoma (SCLC) cell lines and the non-small cell lung carcinoma (NSCLC) cell lines, we studied: (a) production of TGF-β1 and TGF-β2; (b) percentage of cells expressing TGF-β RII; (c) responsiveness of the tumour cell lines to exogenous TGF-β1 or TGF-β2; and (d) presence of mRNA transcripts of the three TGF-β isoforms and of the TGF-β RII. Our results indicate that the SCLC cell lines do not synthesize the isoforms TGF-β1 and TGF-β2 nor the TGF-β RII, thus avoiding inhibitory autocrine and paracrine TGF-β actions. However, NSCLC cell lines express not only TGF-β1, TGF-β2 and TGF-β RII mRNA transcripts, but also synthesize both isoforms and the TGF-β RII. Although approximately 50% of the cells from the studied cell lines expressed the TGF-β RII, different cell lines varied greatly in the sensitivity to the inhibitory action of TGF-β. This could result from alterations in: (i) the structure of TGF-β RII; (ii) the phosphorylation motif of TGF-β RI; (iii) the molecules involved in the intracellular signalling pathway of TGF-β; and (iv) cell cycle regulation.
Repeated allergen challenge in rats increases vitamin A consumption
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Citation Excerpt :
Vitamin A plays an important role in cellular growth and differentiation, and exhibits a wide spectrum of activities, including anti-inflammatory properties. Retinoids can inhibit the respiratory burst and degranulation of stimulated human polymorphonuclear leukocytes probably through the mediation of lipoxygenase products,20 as well as transforming growth factor-β–induced differentiation of tracheal epithelial cells into squamous cells.21 Low serum concentration of vitamin A is reported during acute illness in children with RSV; these low values are associated with more severe illness and are attributed presumably to an increased rate of utilization by the damaged bronchiolar epithelium.3
Vitamin A plays an important role in airways epithelial repair and differentiation. Allergen challenge induces epithelial damage and shedding, which cause an increase in repair activity.
To examine whether repeated allergen challenges could increase vitamin A consumption in a rat model.
Allergic bronchitis was induced in 12 animals, and 12 rats remained naive. After 14 days, repeated allergen inhalation challenges were performed in the sensitized rats for 2 weeks. On day 16, allergen challenge was performed and bronchoconstriction was measured in all 24 rats. On day 30, all rats were killed. BAL was performed and ex vivo tumor necrosis factor (TNF)-α and nitric oxide (NO) production was measured in the lavage cells. Liver, lung tissue, and serum were collected for measurement of vitamin A concentration.
The study rats showed severe bronchoconstriction after allergen challenge compared to the naive rats, and ex vivo TNF-α and NO production was significantly higher in the sensitized rats. Serum and lung concentrations of vitamin A were not different among the two groups. However, the vitamin A liver concentration in the study rats was significantly lower compared to the naive rats.
We conclude that vitamin A utilization is increased during repeated allergen challenge and allergic bronchitis, most probably due to increased demand for epithelial repair.
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Original articleRegulation of proliferation and differentiation of respiratory tract epithelial cells by TGFβ

Abstract

Differentiation

J biol chem

J biol chem

J biol chem

Adv cancer res

Retinoids, differentiation and disease

Biochem soc transactions

Cancer—a problem of developmental biology

New Eng j med

Original article
Regulation of proliferation and differentiation of respiratory tract epithelial cells by TGFβ